Production of bulk amounts of universal RNA for DNA microarrays

In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.     

Evanescent resonator chips: a universal platform with superior sensitivity for fluorescence-based microarrays

In the present paper, we introduce for the first time a novel generation of a universal fluorescence transducer, the so-called evanescent resonator (ER) platform. The device comprises a transparent substrate and a thin dielectric surface layer containing sub-micron corrugated structures. The ER chip exhibits an inherent physical signal amplification due to confinement of excitation energy in the thin surface layer. Energy confinement is based on interference effects created by the abnormal reflection geometry and leads to efficient excitation of surface-bound fluorophores in the evanescent field of the chip. The evanescent resonator platform has the potential to increase the fluorescence yield of labelled biomolecules to more than 100-fold when compared with conventional microarray chips. The new ER device has been developed for analysis of nucleic acids from different species. However, it can be used with all kinds of biomolecular affinity systems. The platform combines superior sensitivity with exceptional reproducibility and ease of use. The chips are compatible with commercially available laser scanners, confocal microscopes, and portable or miniaturised CCD read-out equipment.     

A universal platform for sensitive and selective colorimetric DNA detection based on Exo III assisted signal amplification

Rapid growth of available sequence data has made the detection of nucleic acids critical to the development of modern life sciences. Many amplification methods based on gold nanoparticles and endonuclease for sensitive DNA detection have been developed. However, these approaches require specific target sequence for endonuclease recognition, which cannot be fulfilled in all systems. Replacing the restriction enzyme with a nuclease that does not require any specific recognition sequence may offer a universally adaptable system. Here we have developed a novel homogeneous, colorimetric DNA detection method, which consists of Exo III, a linker DNA, and two DNA-modified gold nanoparticles. This system is simple, low-cost, sensitive and selective. By coupling cyclic enzymatic cleavage and gold nanoparticle for signal amplification, our system provides a colorimetric detection limit of 15 pM, which is 3 orders of magnitude more sensitive than that of a general three-component sandwich assay format. Due to the intrinsic property of Exo III, our method shows excellent detection selectivity for single-base discrimination. More importantly, superior to other methods based on nicking and FokI endonuclease, our target sequence-independent platform is generally applicable for DNA sensing. This new approach could be widely applied to sensitive nucleic acids detection.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

Limited efficiency of universal mini-barcode primers for DNA amplification from desert reptiles, birds and mammals

In recent years, DNA barcoding has emerged as a powerful tool for species identification. We report an extended validation of a universal DNA mini-barcode for amplification of 130-bp COI segments from 23 specimens collected from a desert environment, including 11 reptiles, five mammals and seven birds. Besides the standard double-annealing protocol, we also tested a more stringent single-annealing protocol. The PCR success rate for the amplification of the mini-barcode region was: mammals (4/5), reptiles (5/11) and birds (4/7). These findings demonstrate the limited utility of universal primers for mini-barcoding, at least for these vertebrate taxa that we collected from the Saudi Arabian desert.     

Resin tissue microarrays: a universal format for immunohistochemistry.

Tissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format. Freshly collected tissue fixed in -20C acetone or 10% neutral buffered formaldehyde were cored and arrayed into an intermediary medium of 2% agarose before infiltration of the agarose array with glycol methacrylate resin. Acetone-fixed resin TMA demonstrated improved morphology over acetone-fixed frozen TMA, with no loss of antigenicity. Staining for extracellular, cell surface, and nuclear antigens could be realized with monoclonal and polyclonal antibodies as well as with monomeric single-chain Fv preparations. In addition, when compared with FFPE TMA, formalin-fixed tissue in a resin TMA gave enhanced morphology and subcellular detail. Therefore, resin provides a universal format for the construction of TMAs, providing improved tissue morphology while retaining antigenicity, allows thin-section preparation, and could be used to replace preparation of frozen and FFPE TMAs for freshly collected tissue.     

nuID: a universal naming scheme of oligonucleotides for Illumina, Affymetrix, and other microarrays

Background  

Oligonucleotide probes that are sequence identical may have different identifiers between manufacturers and even between different versions of the same company's microarray; and sometimes the same identifier is reused and represents a completely different oligonucleotide, resulting in ambiguity and potentially mis-identification of the genes hybridizing to that probe.     

RNA synthesis: a requirement for hormone-induced DNA amplification in Rhynchosciara americana

Injection of beta-ecdysone into mid fourth instar larvae of Rhynchosciara americana induced within 23-28 hours after injection a rise in the percentage of 3H-thymidine (3H-TdR) incorporating nuclei in salivary gland region S1 from about 10-20% in the controls to 80-90% in the injected larvae. The 3H-TdR incorporating nuclei displayed a weak continuous labeling pattern or a band-labeling pattern with grains over the vast majority of the bands. The majority of nuclei with a band labeling pattern displayed DNA amplification at the DNA-puff regions.--Injection of actinomycin D at different times after ecdysone injection abolished the higher incorporation rate at the amplifying regions within 15 hours after the injection. However, the percentage of nuclei incorporating 3H-TdR and the frequency of the two labeling patterns remained essentially the same when RNA synthesis was inhibited. Only the over-all rate of 3H-TdR incorporation seemed to be reduced.--These data suggest that in the DNA puff regions the rate of DNA chain elongation is higher when amplification occurs than during a normal replication cycle. It, further, seems that the higher rate during amplification is dependent upon de novo RNA synthesis.     

Statistical issues with microarrays: processing and analysis

The study of gene expression with printed arrays and prefabricated chips is evolving from a qualitative to a quantitative science. Statistical procedures for determining quality control, differential expression, and reproducibility of findings are a natural consequence of this evolution. However, problems inherent to the technologies have raised important issues of how to apply adequate statistical tests. As a consequence, statistical approaches to microarray research are not yet as routine as they are in other sciences. Statistical methods, tailored to microarrays, continue to be adapted and developed. We present an overview of these methods and of outstanding issues in their use and validation.     

Two-color,rolling-circle amplification on antibody microarrays for sensitive,multiplexed serum-protein measurements

The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications.     

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters ΔH°, ΔS° and ΔG° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.     

Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.     

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA

Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 µg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA. gene expression microarray analysis; microdissection; nucleic acid amplification techniques